Fos expression at the cerebellum following non-contact arousal and mating behavior in male rats

Abstract

The cerebellum is considered a center underlying fine movements, cognition, memory and sexual responses. The latter feature led us to correlate sexual arousal and copulation in male rats with neural activity at the cerebellar cortex. Two behavioral paradigms were used in this investigation: the stimulation of males by distant receptive females (non-contact sexual stimulation), and the execution of up to three consecutive ejaculations. The vermis area of the cerebellum was removed following behavioral experiments, cut into sagittal sections, and analyzed with Fos immunohistochemistry to determine neuronal activation. At the mid-vermis region (sections from the midline to 0.1 mm laterally), non-contact stimulation significantly increased the activity of granule neurons. The number of activated cells increased in every lobule, but lobules 1 and 6 to 9 showed the greatest increment. In sexual behavior tests, males reaching one ejaculation had a high number of activated neurons similar to those counted after non-contact stimulation. However, two or three consecutive ejaculations showed a smaller number of Fos-ir cells. In contrast to the mid-vermis region, sections farthest from the midline (0.1 to 0.9 mm laterally) revealed that only lobule 7 expressed activated neurons. These data suggest that a well-delineated group of granule neurons have a sexual biphasic response at the cerebellar vermis, and that Fos in them is under an active degradation mechanism. Thus, they participate as a neural substrate for male rat sexual responses with an activation-deactivation process corresponding with the sensory stimulation and motor performance occurring during copulation.

Figure 1

Cerebellar vermis and cells showing Fos-ir after non-contact sexual stimulation. Top Left: sagittal section of the mid-vermis showing numerical nomenclature of lobules; square in Lobule 1 indicates the amplification area for bottom row micrographs. Top Right: amplification of a lobule to identify the three layers of the cerebellar cortex. The molecular layer (ML), the Purkinje Perikarya layer (PL), and the granule cell layer (GL); inner region shows white matter (wm). This cortical organization is maintained in all lobules. Bottom row: micrographs showing an amplified cerebellar lobule of a non-contact male with a non-receptive female (Ctrl) and 30-min non-contact stimulated male with a receptive female (NCS). An increased number of Fos-ir neurons at the granule cell layer was observed in NCS subjects. Bar = 100 μm.

Figure 2

Number of Fos-ir cells after non-contact stimulation. The graph represents the number of Fos-ir cells in each lobule at 0.1-mm sections. Every lobule showed a significant increase in the number of active neurons after non-contact stimulation with a receptive female (NCS) when compared to its respective control value after non-contact with a non-receptive female (Ctrl). However, lobules 1 and 6 to 9 showed the greatest number of cells. Each point represents the mean ± SEM. * = p<0.05, ** = p<0.01.

Figure 3

Number of Fos-ir cells after non-contact stimulation. 3-D graph showing the number of activated granule cells after non-contact stimulation. The graph shows the number of cells per lobule at different distances from the midline. Lobule 7 possessed the highest number of active cells for lateral areas of the vermis region.

Figure 4

Fos-ir cells after sexual behavior. The sexual behavior of males increased the number of active neurons after one ejaculation (1E) relative to experienced males that remained in the arena alone (Ctrl). The second (2E) and third (3E) consecutive ejaculations also increased the number of activated cells, but to a lesser degree when compared to the increase after one ejaculation. Bar = 100 μm.

Figure 5

Number of Fos-ir cells after sexual behavior. The graph represents the number of Fos-ir cells in each lobule at 0.1-mm sections. Every lobule showed a significant increase in the number of active neurons after one ejaculation (E1) when compared to experienced males that remained in the arena alone (Ctrl). The second (2E) consecutive ejaculation produced a smaller significant increase in the number of active neurons, while the third (3E) consecutive ejaculation produced an increase that was not significantly different from Ctrl values. Each point represents the mean; SEM values were removed for clarity. * = p<0.05, ** = p<0.01.

Figure 6

The sexual vermis. The drawing represents the dorsal view of the vermis area, showing the surface of the lobules that can be seen after removal of the skull, i.e., lobules 6 (L6) to 9 (L9). The topographic shadow represents areas that were activated during non-contact stimulation and sexual behavior. Bar below indicates the average number of neurons counted per region. Lobule 7 expressed the greatest number of Fos-ir neurons around midline and in the whole width of the cerebellar vermis.